title: On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis.
abstract: An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Wang
authors.forename: Junfeng
authors.initials: J
authors.lastname: Ma
authors.forename: Shuaibin
authors.initials: S
authors.lastname: Wu
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D008894:Milk Proteins / Q000032:analysis; D008956:Models, Chemical; D016062:Porosity; D011506:Proteins / Q000032:analysis*; D018892:Proton-Motive Force; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D008894:Milk Proteins; D011506:Proteins; D012259:Ribonuclease, Pancreatic